Abstracts of the 29th Annual Symposium of The Protein Society

2015 Protein Science  
Biological systems have evolved several unique mechanisms to produce inorganic nanomaterials of commercial interest. Furthermore, bio-based methods for nanomaterial synthesis are inherently "green", enabling low-cost and scalable production of nanomaterials under benign conditions in aqueous solutions. However, achieving regulated control of the biological processes necessary for reproducible, scalable biosynthesis of nanomaterials remains a central challenge. This is especially true of quantum
more » ... dots (QDs), which are nanocrystals made from seminconducting metals whose diameter is smaller than the size of its exciton Bohr radius, leading to size-dependent changes in their optical properties. Several studies have described production of QDs from biological systems, but without control over particle size or composition. In this work, we describe the isolation, selection and characterization of a bacterial system capable of regulated, extracellular biosynthesis of metal sulfide QDs with extrinsic control over nanocrystal size. Using directed evolution, we isolated and engineered a bacterial strain (SMCD1) to (1) exhibit enhanced tolerance against aqueous cadmium acetate (2) produce soluble, extracellular nanocrystals and (3) regulate nanocrystal size by varying growth conditions. We estimate yields on the order of grams per liter from batch cultures under optimized conditions, and are able to reproduce the entire size range of CdS QDs described in literature. Furthermore, we are able to generalize this approach to not only cadmium, but PbS QDs as well. Investigation of purified QDs using ESI-MS reveals several putative proteins that may be involved in biosynthesis, and current work is aimed at improving photoluminescent properties as well as long-term aqueous stability. Nonetheless, our approach clearly demonstrates the ability of biological systems to produce advanced, functional nanomaterials, and provides a template for engineering biological systems to high-value materials such as QDs at cost and scale. Proteolysis is a fundamental process in biology; it plays a crucial role across development of multicellular organisms, aids in maintaining tissue homeostasis, and is integral in cell signaling. Intracellular proteolysis frequently focuses on proteasome mediated protein degradation, however the tightly regulated and selective proteolysis mediated by the cysteine-aspartyl specific proteases, caspases, leave their substrates intact. The growing list of caspase substrates now tops 1500 proteins; a key unmet question is to differentiate how individual substrate cleavages directly lead to the profound morphological transformations that are the hallmark of apoptotic cells. We employ an optimized site-specific and inducible split-protein protease to examine the role of a classic apoptotic node, the Caspase Activated DNase (CAD). We describe our engineering platform of post-transcriptional gene replacement (PTGR), whereby endogenous bi-allelic ICAD is knocked down and simultaneously replaced with an engineered allele that is susceptible to cleavage by our engineered TEV protease. Remarkably, selective activation of CAD alone does not induce cell death, although hallmarks of DNA damage are detected in human cancer cell lines. Additionally, we show the utility of our technology in deciphering synthetic lethality resulting from coordinated proteolysis of caspase substrates that control the apoptotic hallmark of chromatin fragmentation. Increasing energy needs have accelerated the demand for renewable alternatives to petroleum-based fuels; engineered microbes for the production of biofuels have the potential to fulfill these energy needs. Fatty acids are the immediate precursors to the advanced biofuels fatty acid methyl esters (FAMEs), which can serve as a "drop in" replacement for D2 diesel. FAMEs derived from medium-chain fatty acids (C8-C12) have been shown to have better cold properties than traditional FAMEs (C16-C22). Here, we engineer a yeast strain for the production of medium chain fatty acids by screening different thioesterases. Our next goal is to couple a medium-chain fatty acid producing yeast strain to our previously developed medium-chain fatty acid GPCR-based sensor, in order to engineer a yeast strain with improved medium-chain fatty acid production via directed evolution. Nuclear magnetic resonance (NMR) is a powerful biophysical method for studying protein-ligand interactions in solution and elucidating the mechanism of action of potential inhibitors. However, protein NMR can be complicated by the overlap of 1H and other resonances, hence the resolution needed to assign spectra precisely can be hard to achieve [1]. 19F-NMR is increasingly being used to study conformational changes and protein-ligand interactions in solution because 19F is (i) a spin 1 = 2 nucleus, (ii) 100% naturally abundant, (iii) 83% as sensitive to NMR detection as 1H, (iv) not present in most biological systems, and (v) its chemical shift is particularly sensitive to changes in local environment [2]. Recent advancements in NMR instrument and probe design have made 19F-NMR more sensitive and more widely available; consequently, 19F-NMR is finding growing application in research. Here, we report the use of 19F-NMR to study two biomedically important protein systems. Proteins can be fluorine-labelled either by biological incorporation of fluorinated amino acids or by site-specific chemical ligation [3]. 3-bromo-1,1,1-trifluoroacetone (BTFA) has been developed as a useful reagent for importing fluorine into proteins via nucleophilic substitution such as with a cysteinyl-thiol [4]. The São Paulo metallo-b-lactamase-1 (SPM-1), a B1 sub-family metallo-b-lactamase, containing only one cysteine (Cys221) coordinating the second Zn(II) cation in its active site, was 19F-labelled using BTFA. The interactions of SPM-1 with various potential inhibitors were reported by 19F-NMR, which enabled monitoring SPM-1 conformational changes on ligand binding and informed on binding strength by enabling KD measurements. In a second study, prolyl hydroxylase 2 (PHD2), an enzyme involved in human oxygen sensing, was 19F-labelled and its interactions with its co-substrate, 2-oxoglutarate (2OG), and peptide substrate (CODD) were monitored by 19F-NMR. Conformational changes of PHD2 on 2OG and CODD binding were consistent with crystallographic analyses. Finally, 19F-NMR was used to study solvent exposure of the complex and its dynamics in solution through relaxation dispersion of the 19F-nucleus at different temperatures. Overall, the results illustrate the power of 19F-NMR for monitoring ligand binding and conformational changes.
doi:10.1002/pro.2823 pmid:26452528 pmcid:PMC4632850 fatcat:ejmksoqj3zbsrkabl4jyp7emgi